Development of molecular methods that enable production of infectious virus from DNA copies of their genomes has significantly improved our knowledge of RNA virus life cycles and pathogenesis, by permitting the development of “reverse genetics”, i.e., studies of the impact of specific mutations on the biological properties of viruses.
However, current methodologies for construction of infectious cDNA clones are unpredictable and laborious processes frequently associated with undesirable mutations or unstable/toxic clones in bacteria.
This has spawned great interest in alternative methods for generating RNA virus. Various methodological improvements, such as the use of alternative hosts, low-copy-number plasmids, cosmid vectors, bacterial artificial chromosomes, modified promoters or modified viral genome sequences with reduced cryptic bacterial promoter activity have been proposed.
Bacterium-free approaches were also developed for example with Tick-borne encephalitis (TBEV) by Gritsun and Gould in 1995 and with West Nile virus (WNV) and Dengue virus (DENV) by Edmonds et al. and Siridechadilok et al., respectively, in 2013.
Although they represented significant advances, these methods require substantial optimisation for each virus studied and do not provide a unified methodological process.
There is thus a long felt unfulfilled need for an alternative method for generating an infectious RNA virus, which is efficient, precise and prompt.